Compositions and methods for modulating hemostasis using variant forms of activated factor v

ABSTRACT

Methods for the treatment of coagulation disorders using Factor V/Va variants are provided.

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 60/776,124 filed Feb. 23, 2006, the entirecontents being incorporated by reference herein as though set forth infull.

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Number K01 DK60580-01 and P01-HL74124-01 (Project 2).

FIELD OF THE INVENTION

The present invention relates the fields of medicine and hematology.More specifically, the invention describes therapeutic strategies usingactivated forms of FV and derivatives thereof for modulating thecoagulation cascade in patients in need thereof.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Hemophilia is an X-linked hemorrhagic disorder resulting from mutationsin either the FVIII (hemophilia A) or FIX (hemophilia B) genes, with anincidence of approximately 1:5,000 male individuals worldwide. Affectedindividuals commonly present spontaneous hemorrhages and prolongedbleeding after trauma or surgery. Severely affected subjects presentFVIII or FIX levels lower than 1% of normal and comprise the majority ofclinically symptomatic cases. The remaining patients have mild tomoderate disease with factor levels of 1-30%[1].

The clinical presentation of hemophilia is essentially indistinguishablefor FVIII or FIX deficiency. However, there is clear evidence that theclinical phenotype of hemophilia varies among patients with similarresidual factor levels or even with the same underlying mutation [2-4].Therefore, it is possible that other genetic or acquired factorsinfluence the hemophilia phenotype. The current understanding of thegenetic basis of venous thrombosis provides an opportunity to determinewhether these risk factors could improve the hemophilia phenotype.

Thrombin generation is in part controlled by activated protein C (APC),which is formed by limited proteolysis of the zymogen protein C by thethrombin-thrombomodulin complex. The anticoagulant effect of APC resultsfrom the inactivation of both factors Va and VIIIa on membranesurfaces[5]. The most common inherited thrombophilia results from amutation in the FV gene (Arg 506 to Gln) known as FV Leiden (FVL).Because Arg 506 is the initial cleavage site for APC, FVL is inactivatedat approximately one tenth the rate of normal FVa [6], which result inhigh thrombin levels that create a procoagulant state.

FVL is the most commonly investigated modifier of the hemophiliaphenotype because it is present in 2-5% of the Caucasian population[7,8]. Initial reports suggested the amelioration of the severehemophilia A phenotype among subjects with FVL [9,10]. Further studies,however, failed to demonstrate the clinical impact of such association.In screening over 700 hemophilia subjects, 35 cases of FVL have beenidentified. In only half of these cases (14 hemophilia A and 1hemophilia B) the association was considered beneficial in terms offrequency of bleeds and/or factor consumption over time [9-14]. Thereasons for these discrepancies are not clear but could relate to thesmall number of subjects, differences in age groups, the presence ofunderlying infectious diseases, and the retrospective nature of thestudy. The results of a pediatric study have been informative in thismatter since many of the complications common among adults are notconfounding factors in children. This case-control study demonstratedthat among hemophilia A children with FVL or with other thrombophiliarisk factors, the onset of the first bleeding episode was delayed [14].

There is also in vitro evidence that the FVL mutation can modifythrombin generation in FVIII [15] or FIX deficient plasma [16].Moreover, the assessment of the fibrinolytic system in hemophiliarevealed that thrombin-induced clots in FVIII or FIX deficient plasmawere lysed prematurely [17]. Therefore, the enhanced generation ofthrombin by FVL may also increase the resistance of the fibrin clot topremature lysis [18,19].

SUMMARY OF THE INVENTION

To gain insight into the discrepancies between clinical and laboratorialassessments of the impact of FVL on hemophilia, we took advantage of theavailability of genetically engineered mice for severe hemophilia andFVL. These murine models provide the opportunity to address the role ofthis thrombotic risk factor in modifying the hemophiliac phenotype invivo with minimal influence from environmental factors. Using theseunique model systems in conjunction with biochemical assays andreal-time imaging of clot formation in living animals, we have foundthat activated forms of FV can beneficially modify the hemophiliaphenotype.

A pharmaceutical composition comprising the activated forms of FV aswell as cleavage resistant forms of activated FV of the invention in abiologically compatible carrier which may be directly infused into apatient is also provided. Another preferred aspect of the inventionincludes methods for the treatment of a hemostasis related disorder in apatient in need thereof comprising administration of a therapeuticallyeffective amount of the activated forms of FV containing pharmaceuticalcompositions described herein. Such methods should have efficacy in thetreatment of disorders where a pro-coagulant is needed and include,without limitation, hemophilia A and B, hemophilia A and B associatedwith inhibitory antibodies, coagulation factor deficiency, vitamin Kepoxide reductase C1 deficiency, gamma-carboxylase deficiency, bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation treatment disorders, Bernard Soulier syndrome,Glanzman thromblastemia, and storage pool deficiency.

Another aspect of the invention, includes host cells expressing thevariant activated forms of FV of the invention in order to produce largequantities thereof. Methods for isolating and purifying the activatedforms of FV are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Clotting assays in murine models of hemophilia or Factor VLeiden (FVL) and in normal controls. Panels A, C, and E: Modified onestage activated partial thromboplastin assay (aPTT) in mouse plasma.Panels B, D and F: Thrombin-antithrombin complex (TAT) levels in miceplasma. The numbers of animals per group are indicated. Adult hemophiliaA (HA) or hemophilia B (HB) mice without FVL were compared with animalsheterozygous (+/−) or homozygous (+/+) for FVL. P values were calculatedby ANOVA. NS: not significant.

FIG. 2. Hemostatic assessment following tail clip assay in hemophilicmice crossed with Factor V Leiden (FVL). Blood loss was measured byhemoglobin content of the saline solution by optical density at A₅₇₅post tail clipping of hemophilia mice. Panel A: Hemophilia A (HA) mice,HA heterozygous (+/−) and homozygous for FVL (+/+) were compared. PanelB: Littermates hemophilia B (HB), HB heterozygous (+/−) and homozygousfor FVL (+/+) were compared. The numbers of animals per group areindicated. P value was calculated by t-test.

FIG. 3. Platelet deposition in arterial thrombi in mice uponlaser-induced endothelial damage using fluorescence signals. Upperpanels represent data from hemophilia A (HA) mice and bottom panels aredata from hemophilia B (HB) mice. Platelet deposition was monitoredusing an anti-CD41Alexa-555 labeled antibody and composite images at˜120 seconds post laser injury are shown. Thrombus formation wasassessed at the baseline (panel A) and after protein replacement (panelB). Hemophilic mice homozygous or heterozygous for FVL are shown inpanels D to F. Hemophilic mice injected with 30 μg of FVa (Panel C).

FIG. 4. Time course of platelet accumulation in arterial thrombi.Platelet deposition in developing thrombin over time was monitored withanti-CD41 Alexa-55 labeled antibody. The arbitrary relative fluorescentunit (RFU) represents the median platelet-derived fluorescence forseveral thrombi. Panel A represents hemophilia B mice (HB), HB miceinfused with human F.IX concentrated and hemostatically normal C57B1/6mice as control (CT). Panel B represents hemophilia A (HA) or KB micehomozygous for Factor V Leiden (FVL) and naïve hemophilic mice. Panels Cand D represents hemophilia A (HA) or (HB) naïve mice or injected withpurified FV or FVa proteins. These experiments are representative ofmultiple thrombi (>5 thrombi/mouse) in (2-4) mice per group, as shown intable 2.

FIG. 5 Schematic representation of FV, FV-810 and FVa. The sequenceabove FV-810 indicates which B-domain elements have been deleted. IIacleavage sites are indicated as well as the molecular weight of thevarious fragments.

DETAILED DESCRIPTION OF THE INVENTION

The role of factor V Leiden (FVL) as a modifier of the severe hemophiliaphenotype is still unclear. We used mice with hemophilia A or B crossedwith FVL to elucidate in vivo parameters of hemostasis. Real-timethrombus formation in the microcirculation was monitored by depositionof labeled platelets upon laser-induced endothelial injury usingwidefield microscopy in living animals. No thrombi formed in hemophilicA or B mice following vascular injuries. However, hemophilic mice,either heterozygous or homozygous for FVL, formed clots at all injuredsites. Injection of purified activated FV into hemophilic A or B micecould mimic the in vivo effect of FVL. In contrast to these responses toa laser injury in a microvascular bed, FVL did not provide sustainedhemostasis following damage of large vessels in a ferric chloridecarotid artery injury model, despite of the improvement of clottingtimes and high circulating thrombin levels. Together these data provideevidence that FVL has the ability to improve the hemophilia A or Bphenotype, but this effect is principally evident at themicrocirculation level following a particular vascular injury. Ourobservations may partly explain the heterogeneous clinical evidence ofthe beneficial role of FVL in hemophilia.

Thus, in accordance with one aspect of the invention, methods for thetreatment of hemophilia are provided. An exemplary method entailsadministration of an effective amount of an activated form of FV or aderivative thereof to patient to enhance clot formation, therebyameliorating the symptoms of hemophilia. This treatment initiatescoagulation in individuals who lack intrinsic pathway proteins, haveinhibitors to these proteins, or who have some other hemostaticabnormality which would benefit by administration of an activated formof FV. Administration of active FV or engineered derivatives of anactivated form of FV which have the activated form of FV-like propertiesto by-pass deficiencies in the intrinsic or extrinsic pathway isdisclosed.

Protein replacement therapy using recombinant or plasma-derived forms offactor VIII (or B-domain deleted FVIII), factor IX, or factor VIIa(NovoSeven) is currently the mainstay of hemophilia care. While thistreatment regime has limitations, it is very effective and has helpedthousands of patients. Over the past 20 years significant progress hasbeen made by several groups in understanding the biochemistry of FV.Factor V circulates in plasma as a single chain procofactor at aconcentration of 7 μg/mL (20 nM) and has a half-life of ˜12 hours. It isa large (M_(r)=330,000, 2196 amino acids) heavily glycosylated, singlechain, multi-domain (A1-A2-B-A3-C1-C2) protein which is synthesized inthe liver and is homologous to factor VII. Factor V is secreted as aninactive procofactor and cannot function in the prothrombinase complex(FXa, FVa, anionic membranes, and calcium). This is consistent with theobservation that FV binds very weakly, if at all, to FXa andprothrombin, and indicates that proteolytic conversion of FV to FValeads to appropriate structural changes which impart cofactor function.

Thrombin is established as the most robust activator of FV. Proteolysisoccurs at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵ generating FVa_(IIa), aheterodimer composed of an N-terminal 105 kDa heavy chain associated viaCa²⁺ ions to the C-terminal 74/71 kDa light chain. See FIG. 5. Thelarge, heavily glycosylated B domain, spanning amino acids 710-1545, isnot necessary for cofactor activity and is released during activation.

In addition to using activated factor V (FVa), we have engineered singlechain FV derivatives in which large segments of the B-domain have beendeleted (For example, FVdes⁸¹¹⁻¹⁴⁹¹; FV-810, Journal of BiologicalChemistry, (2004) 279: 21643-21650; FIG. 5). We have also madeadditional derivatives which have cofactor-like properties as well. Forexample, FV-902 (factor V des903-1491) also has activity profiles thatare similar to FV-810 and the activated form of FV. These B-domaintruncated derivatives exhibit functional proprieties equivalent to FVa,even in the absence of intentional proteolysis. The usefulness of thesederivatives in the context of this application are: 1) they are secretedfrom the mammalian cell line in a single-chain form, and do not requireintentional proteolytic activation with thrombin or factor Xa; 2) theyhave activities that are comparable to two-chain active factor Va; 3)they do not need to be further processed with thrombin; and 4) they maybe more stable in plasma compared to two-chain factor Va (i.e havebetter half-lives).

Additional Useful Derivatives Include:

FV-810; factor V lacking amino acids 811-1491; (published JBC, 279,2004, 21643-21650)FV-859; factor V lacking amino acids 860-1491;FV-866; factor V lacking amino acids 867-1491;FV-902; factor V lacking amino acids 903-1491;FV-924; factor V lacking amino acids 923-1491;FV-937; factor V lacking amino acids 938-1491;FV-956; factor V lacking amino acids 957-1491;

Others Include:

FV-1033-B58-s131; factor V lacking amino acids 1034-1491 with aminoacids 900-1030 exchanged with amino acids 907-1037 of factor VIII;FV-1033-B58-s104; factor V lacking amino acids 1034-1491 with aminoacids 904-1007 exchanged with amino acids 972-1075 of factor VIII;FV-1033-B58-s46; factor V lacking amino acids 1034-1491 with amino acids963-1008 exchanged with amino acids 1032-1077 of factor VIII;Each of the above derivatives exhibit functional properties which arecomparable to two chain the activated form of FV.

In order to enhance stability of the molecules in vivo and provideresistance or protection the protein C pathway each of the abovederivatives could be modified at:

Arg506

Arg306

Arg679

While in theory any amino acid could be exchanged at this site, Gln ispreferable.

Thus, the methods and compositions of the invention could be used totreat patients with hemophilia A and hemophilia B; patients withhemophilia A and B who have inhibitory antibodies to either factor VIIIor factor IX, respectively; and other hemostatic disorders.

I. DEFINITIONS

Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specification andclaims.

The phrase “activated form of FV” refers to a modified form of FV whichhas been genetically altered such that it exhibits cofactor activitycomparable to two-chain FVa in the absence of intentional proteolysisand in the absence of specific cofactors. Preferred sites for amino acidalterations in the parent FV molecule are described above.

The phrase “hemostasis related disorder” refers to bleeding disorderssuch as hemophilia A and B, hemophilia A and B patients with inhibitoryantibodies, deficiencies in coagulation Factors, VII, IX and X, XI, V,XII, II, von Willebrand factor, combined FV/FVIII deficiency, vitamin Kepoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation associated with heparin, low molecular weightheparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e.FXa inhibitors); and platelet disorders such as, Bernard Souliersyndrome, Glanzman thromblastemia, and storage pool deficiency.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it originates. For example,the “isolated nucleic acid” may comprise a DNA or cDNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into theDNA of a prokaryote or eukaryote.

With respect to RNA molecules of the invention, the term “isolatednucleic acid” primarily refers to an RNA molecule encoded by an isolatedDNA molecule as defined above. Alternatively, the term may refer to anRNA molecule that has been sufficiently separated from RNA moleculeswith which it would be associated in its natural state (i.e., in cellsor tissues), such that it exists in a “substantially pure” form (theterm “substantially pure” is defined below).

With respect to protein, the term “isolated protein” or “isolated andpurified protein” is sometimes used herein. This term refers primarilyto a protein produced by expression of an isolated nucleic acid moleculeof the invention. Alternatively, this term may refer to a protein whichhas been sufficiently separated from other proteins with which it wouldnaturally be associated, so as to exist in “substantially pure” form.

The term “promoter region” refers to the transcriptional regulatoryregions of a gene, which may be found at the 5′ or 3′ side of the codingregion, or within the coding region, or within introns.

The term “vector” refers to a small carrier DNA molecule into which aDNA sequence can be inserted for introduction into a host cell where itwill be replicated.

An “expression vector” is a specialized vector that contains a gene ornucleic acid sequence with the necessary regulatory regions needed forexpression in a host cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of a coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector. This definition is alsosometimes applied to the arrangement of nucleic acid sequences of afirst and a second nucleic acid molecule wherein a hybrid nucleic acidmolecule is generated.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-99% by weight,of the compound of interest. Purity is measured by methods appropriatefor the compound of interest (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

The phrase “consisting essentially of” when referring to a particularnucleotide sequence or amino acid sequence means a sequence having theproperties of a given SEQ ID NO:. For example, when used in reference toan amino acid sequence, the phrase includes the sequence per se andmolecular modifications that would not affect the basic and novelcharacteristics of the sequence.

The term “oligonucleotide,” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo- or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application for which the oligonucleotideis used.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and method of use. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides.

The probes herein are selected to be “substantially” complementary todifferent strands of a particular target nucleic acid sequence. Thismeans that the probes must be sufficiently complementary so as to beable to “specifically hybridize” or anneal with their respective targetstrands under a set of pre-determined conditions. Therefore, the probesequence need not reflect the exact complementary sequence of thetarget. For example, a non-complementary nucleotide fragment may beattached to the 5 or 3′ end of the probe, with the remainder of theprobe sequence being complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to act functionally as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product.

The primer may vary in length depending on the particular conditions andrequirements of the application. For example, in diagnosticapplications, the oligonucleotide primer is typically 15-25 or morenucleotides in length. The primer must be of sufficient complementarityto the desired template to prime the synthesis of the desired extensionproduct, that is, to be able to anneal with the desired template strandin a manner sufficient to provide the 3′ hydroxyl moiety of the primerin appropriate juxtaposition for use in the initiation of synthesis by apolymerase or similar enzyme. It is not required that the primersequence represent an exact complement of the desired template. Forexample, a non-complementary nucleotide sequence may be attached to the5′ end of an otherwise complementary primer. Alternatively,non-complementary bases may be interspersed within the oligonucleotideprimer sequence, provided that the primer sequence has sufficientcomplementarity with the sequence of the desired template strand tofunctionally provide a template-primer complex for the synthesis of theextension product.

The term “percent identical” is used herein with reference tocomparisons among nucleic acid or amino acid sequences. Nucleic acid andamino acid sequences are often compared using computer programs thatalign sequences of nucleic or amino acids thus defining the differencesbetween the two. For purposes of this invention comparisons of nucleicacid sequences are performed using the GCG Wisconsin Package version9.1, available from the Genetics Computer Group in Madison, Wis. Forconvenience, the default parameters (gap creation penalty=12, gapextension penalty=4) specified by that program are intended for useherein to compare sequence identity. Alternately, the Blastn 2.0 programprovided by the National Center for Biotechnology Information (athttp://www.ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J Mol Biol215:403-410) using a gapped alignment with default parameters, may beused to determine the level of identity and similarity between nucleicacid sequences and amino acid sequences.

II. PREPARATION OF ACTIVATED FORMS OF FV ENCODING NUCLEIC ACIDMOLECULES, POLYPEPTIDES AND DERIVATIVES THEREOF A. Nucleic AcidMolecules

Nucleic acid molecules encoding the activated forms of FV or derivativethereof of the invention may be prepared by using recombinant DNAtechnology methods. The availability of nucleotide sequence informationenables preparation of isolated nucleic acid molecules of the inventionby a variety of means. For example, nucleic acid sequences encoding theactivated forms of FV or derivative thereof polypeptide may be isolatedfrom appropriate biological sources using standard protocols well knownin the art.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in a plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell. Alternatively, the nucleic acids may be maintained invector suitable for expression in mammalian cells. In cases wherepost-translational modification affects the activated forms of FV orderivative thereof function, it is preferable to express the molecule inmammalian cells.

The activated forms of FV or derivative thereof-encoding nucleic acidmolecules of the invention include cDNA, genomic DNA, RNA, and fragmentsthereof which may be single- or double-stranded. Thus, this inventionprovides oligonucleotides (sense or antisense strands of DNA or RNA)having sequences capable of hybridizing with at least one sequence of anucleic acid molecule of the present invention. Such oligonucleotidesare useful as probes for detecting the activated form of FV expression.

B. Proteins

A full-length activated form of FV or derivative thereof polypeptide ofthe present invention may be prepared in a variety of ways, according toknown methods. The protein may be purified from appropriate sources,e.g., transformed bacterial or animal cultured cells or tissues whichexpress the activated form of FV, by immunoaffinity purification.However, this is not a preferred method due to the low amount of proteinlikely to be present in a given cell type at any time.

The availability of nucleic acid molecules encoding an activated form ofFV or derivative thereof polypeptide enables production of the activatedform of FV or derivative thereof using in vitro expression methods knownin the art. For example, a cDNA or gene may be cloned into anappropriate in vitro transcription vector, such as pSP64 or pSP65 for invitro transcription, followed by cell-free translation in a suitablecell-free translation system, such as wheat germ or rabbit reticulocytelysates. In vitro transcription and translation systems are commerciallyavailable, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville,Md.

Alternatively, according to a preferred embodiment, larger quantities ofthe activated form of FV or derivative thereof may be produced byexpression in a suitable prokaryotic or eukaryotic expression system.For example, part or all of a DNA molecule encoding the activated formof FV for example, may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli or a mammalian cell suchas CHO or Hela cells. Alternatively, in a preferred embodiment, taggedfusion proteins comprising the activated form of FV or derivativethereof can be generated. Such activated form of FV or derivativethereof-tagged fusion proteins are encoded by part or all of a DNAmolecule, ligated in the correct codon reading frame to a nucleotidesequence encoding a portion or all of a desired polypeptide tag which isinserted into a plasmid vector adapted for expression in a bacterialcell, such as E. coli or a eukaryotic cell, such as, but not limited to,yeast and mammalian cells. Vectors such as those described abovecomprise the regulatory elements necessary for expression of the DNA inthe host cell positioned in such a manner as to permit expression of theDNA in the host cell. Such regulatory elements required for expressioninclude, but are not limited to, promoter sequences, transcriptioninitiation sequences, and enhancer sequences.

The activated form of FV or derivative thereof proteins, produced bygene expression in a recombinant prokaryotic or eukaryotic system may bepurified according to methods known in the art. In a preferredembodiment, a commercially available expression/secretion system can beused, whereby the recombinant protein is expressed and thereaftersecreted from the host cell, to be easily purified from the surroundingmedium. If expression/secretion vectors are not used, an alternativeapproach involves purifying the recombinant protein by affinityseparation, such as by immunological interaction with antibodies thatbind specifically to the recombinant protein or nickel columns forisolation of recombinant proteins tagged with 6-8 histidine residues attheir N-terminus or C-terminus. Alternative tags may comprise the FLAGepitope, GST or the hemagglutinin epitope. Such methods are commonlyused by skilled practitioners.

The activated form of FV or derivative thereof proteins, prepared by theaforementioned methods, may be analyzed according to standardprocedures. For example, such proteins may be subjected to amino acidsequence analysis, according to known methods.

As discussed above, a convenient way of producing a polypeptideaccording to the present invention is to express nucleic acid encodingit, by use of the nucleic acid in an expression system. A variety ofexpression systems of utility for the methods of the present inventionare well known to those of skill in the art.

Accordingly, the present invention also encompasses a method of making apolypeptide (as disclosed), the method including expression from nucleicacid encoding the polypeptide (generally nucleic acid). This mayconveniently be achieved by culturing a host cell, containing such avector, under appropriate conditions which cause or allow production ofthe polypeptide. Polypeptides may also be produced in in vitro systems,such as reticulocyte lysate.

III. USES OF THE ACTIVATED FORM OF FV OR DERIVATIVE THEREOF—ENCODINGNUCLEIC ACIDS AND PROTEINS

The activated form of FV polypeptide or derivative thereof or nucleicacids encoding the same having altered coagulation activities may beused according to this invention, for example, as therapeutic and/orprophylactic agents which modulate the blood coagulation cascade. Thepresent inventors have discovered that these molecules can be altered toincrease coagulation.

A. In a preferred embodiment of the present invention, the activatedform of FV or derivative thereof may be administered to a patient viainfusion in a biologically compatible carrier, preferably viaintravenous injection. The activated form of FV or derivative thereof ofthe invention may optionally be encapsulated into liposomes or mixedwith other phospholipids or micelles to increase stability of themolecule. The activated form of FV or derivative thereof may beadministered alone or in combination with other agents known to modulatehemostasis (e.g., Factor VIIa, FIX, FVIII or FX/Xa and derivativesthereof). An appropriate composition in which to deliver the activatedform of FV or derivative thereof may be determined by a medicalpractitioner upon consideration of a variety of physiological variables,including, but not limited to, the patient's condition and hemodynamicstate. A variety of compositions well suited for different applicationsand routes of administration are well known in the art and are describedhereinbelow.

The preparation containing the purified activated forms of FV orderivative thereof contains a physiologically acceptable matrix and ispreferably formulated as a pharmaceutical preparation. The preparationcan be formulated using substantially known prior art methods, it can bemixed with a buffer containing salts, such as NaCl, CaCl₂, and aminoacids, such as glycine and/or lysine, and in a pH range from 6 to 8.Until needed, the purified preparation containing the factor V/Va analogcan be stored in the form of a finished solution or in lyophilized ordeep-frozen form. Preferably the preparation is stored in lyophilizedform and is dissolved into a visually clear solution using anappropriate reconstitution solution.

Alternatively, the preparation according to the present invention canalso be made available as a liquid preparation or as a liquid that isdeep-frozen.

The preparation according to the present invention is especially stable,i.e., it can be allowed to stand in dissolved form for a prolonged timeprior to application.

The preparation according to the present invention can be made availableas a pharmaceutical preparation with the activated form of FV orderivative thereof in the form of a one-component preparation or incombination with other factors in the form of a multi-componentpreparation.

Prior to processing the purified protein into a pharmaceuticalpreparation, the purified protein is subjected to the conventionalquality controls and fashioned into a therapeutic form of presentation.In particular, during the recombinant manufacture, the purifiedpreparation is tested for the absence of cellular nucleic acids as wellas nucleic acids that are derived from the expression vector, preferablyusing a method, such as is described in EP 0 714 987.

Another feature of this invention relates to making available apreparation which contains an activated form of FV or derivative thereofwith a high stability and structural integrity and which, in particular,is free from inactive factor V/Va analog intermediates andautoproteolytic degradation products and which can be produced byactivating a factor V analog of the type described above and byformulating it into an appropriate preparation.

The pharmaceutical preparation may contain dosages of between 10-1000μg/kg, more preferably between about 10-250 μg/kg and most preferablybetween 10 and 75 μg/kg, with 40 μg/kg of the variant factor Vpolypeptide being particularly preferred. Patients may be treatedimmediately upon presentation at the clinic with a bleed. Alternatively,patients may receive a bolus infusion every one to three hours, or ifsufficient improvement is observed, a once daily infusion of theactivated form of FV or derivative thereof described herein.

B. The Activated Form of FV or Derivative Thereof or DerivativeThereof-Encoding Nucleic Acids

The activated form of FV or derivative thereof-encoding nucleic acidsmay be used for a variety of purposes in accordance with the presentinvention. In a preferred embodiment of the invention, a nucleic aciddelivery vehicle (i.e., an expression vector) for modulating bloodcoagulation is provided wherein the expression vector comprises anucleic acid sequence coding the activated form of FV or derivativethereof polypeptide, or a functional fragment thereof as describedherein. Administration of the activated form of FV or derivativethereof-encoding expression vectors to a patient, results in theexpression of the activated form of FV or derivative thereof polypeptidewhich serves to enhance coagulation. In accordance with the presentinvention, the activated form of FV or derivative thereof encodingnucleic acid sequence may encode the activated form of FV or derivativethereof polypeptide as described herein whose expression modulateshemostasis.

Expression vectors comprising the activated form of FV or derivativethereof nucleic acid sequences may be administered alone, or incombination with other molecules useful for modulating hemostasis.According to the present invention, the expression vectors orcombination of therapeutic agents may be administered to the patientalone or in a pharmaceutically acceptable or biologically compatiblecomposition.

In a preferred embodiment of the invention, the expression vectorcomprising nucleic acid sequences encoding the activated form of FV orderivative thereof is a viral vector. Viral vectors which may be used inthe present invention include, but are not limited to, adenoviralvectors (with or without tissue specific promoters/enhancers),adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-2,AAV-5, AAV-7, and AAV-8) and hybrid AAV vectors, lentivirus vectors andpseudo-typed lentivirus vectors [e.g., Ebola virus, vesicular stomatitisvirus (VSV), and feline immunodeficiency virus (FIV)], herpes simplexvirus vectors, vaccinia virus vectors, and retroviral vectors.

In a preferred embodiment of the present invention, methods are providedfor the administration of a viral vector comprising nucleic acidsequences encoding an activated form of FV or derivative thereof, or afunctional fragment thereof. Adenoviral vectors of utility in themethods of the present invention preferably include at least theessential parts of adenoviral vector DNA. As described herein,expression of the activated form of FV or derivative thereof polypeptidefollowing administration of such an adenoviral vector serves to modulatehemostasis.

Recombinant adenoviral vectors have found broad utility for a variety ofgene therapy applications. Their utility for such applications is duelargely to the high efficiency of in vivo gene transfer achieved in avariety of organ contexts.

Adenoviral particles may be used to advantage as vehicles for adequategene delivery. Such virions possess a number of desirable features forsuch applications, including: structural features related to being adouble stranded DNA nonenveloped virus and biological features such as atropism for the human respiratory system and gastrointestinal tract.Moreover, adenoviruses are known to infect a wide variety of cell typesin vivo and in vitro by receptor-mediated endocytosis. Attesting to theoverall safety of adenoviral vectors, infection with adenovirus leads toa minimal disease state in humans comprising mild flu-like symptoms.

Due to their large size (˜36 kilobases), adenoviral genomes are wellsuited for use as gene therapy vehicles because they can accommodate theinsertion of foreign DNA following the removal of adenoviral genesessential for replication and nonessential regions. Such substitutionsrender the viral vector impaired with regard to replicative functionsand infectivity. Of note, adenoviruses have been used as vectors forgene therapy and for expression of heterologous genes.

For a more detailed discussion of the use of adenovirus vectors utilizedfor gene therapy, see Berkner, 1988, Biotechniques 6:616-629 andTrapnell, 1993, Advanced Drug Delivery Reviews 12:185-199.

It is desirable to introduce a vector that can provide, for example,multiple copies of a desired gene and hence greater amounts of theproduct of that gene. Improved adenoviral vectors and methods forproducing these vectors have been described in detail in a number ofreferences, patents, and patent applications, including: Mitani and Kubo(2002, Curr Gene Ther. 2(2); 135-44); Olmsted-Davis et al. (2002, HumGene Ther. 13(11): 1337-47); Reynolds et al. (2001, Nat. Biotechnol.19(9):838-42); U.S. Pat. Nos. 5,998,205 (wherein tumor-specificreplicating vectors comprising multiple DNA copies are provided);6,228,646 (wherein helper-free, totally defective adenovirus vectors aredescribed); 6,093,699 (wherein vectors and methods for gene therapy areprovided); 6,100,242 (wherein a transgene-inserted replication defectiveadenovirus vector was used effectively in in vivo gene therapy ofperipheral vascular disease and heart disease); and International PatentApplication Nos. WO 94/17810 and WO 94/23744.

For some applications, an expression construct may further compriseregulatory elements which serve to drive expression in a particular cellor tissue type. Such regulatory elements are known to those of skill inthe art and discussed in depth in Sambrook et al. (1989) and Ausubel etal. (1992). The incorporation of tissue specific regulatory elements inthe expression constructs of the present invention provides for at leastpartial tissue tropism for the expression of the activated form of FV orderivative thereof or functional fragments thereof. For example, an E1deleted type 5 adenoviral vector comprising nucleic acid sequencesencoding the activated form of FV or derivative thereof under thecontrol of a cytomegalovirus (CMV) promoter may be used to advantage inthe methods of the present invention.

Exemplary Methods for Producing Adenoviral Vectors

Adenoviral vectors for recombinant gene expression have been produced inthe human embryonic kidney cell line 293 (Graham et al., 1977, J. Gen.Virol. 36:59-72). This cell line is permissive for growth of adenovirus2 (Ad2) and adenovirus 5 mutants defective in E1 functions because itcomprises the left end of the adenovirus 5 genome and, therefore,expresses E1 proteins. E1 genes integrated into the cellular genome of293 cells are expressed at levels which facilitate the use of thesecells as an expression system in which to amplify viral vectors fromwhich these genes have been deleted. 293 cells have been usedextensively for the isolation and propagation of E1 mutants, forhelper-independent cloning, and for expression of adenovirus vectors.Expression systems such as the 293 cell line, therefore, provideessential viral functions in trans and thereby enable propagation ofviral vectors in which exogenous nucleic acid sequences have beensubstituted for E1 genes. See Young et al. in The Adenoviruses,Ginsberg, ed., Plenum Press, New York and London (1984), pp. 125-172.

Other expression systems well suited to the propagation of adenoviralvectors are known to those of skill in the art (e.g., HeLa cells) andhave been reviewed elsewhere.

Also included in the present invention is a method for modulatinghemostasis comprising providing cells of an individual with a nucleicacid delivery vehicle encoding the activated form of FV or derivativethereof polypeptide and allowing the cells to grow under conditionswherein the activated form of FV or derivative thereof polypeptide isexpressed.

From the foregoing discussion, it can be seen that the activated form ofFV or derivative thereof polypeptide or nucleic acids encoding the samemay be used in the treatment of disorders associated with aberrant bloodcoagulation.

C. Pharmaceutical Compositions

The expression vectors of the present invention may be incorporated intopharmaceutical compositions that may be delivered to a subject, so as toallow production of a biologically active protein (e.g., the activatedform of FV or derivative thereof polypeptide or functional fragment orderivative thereof). In a particular embodiment of the presentinvention, pharmaceutical compositions comprising sufficient geneticmaterial to enable a recipient to produce a therapeutically effectiveamount of the activated form of FV or derivative thereof polypeptide caninfluence hemostasis in the subject. Alternatively, as discussed above,an effective amount of the variant Factor V polypeptide may be directlyinfused into a patient in need thereof. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agentswhich influence hemostasis.

In preferred embodiments, the pharmaceutical compositions also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce an immune responseharmful to the individual receiving the composition, and which may beadministered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, liquids such as water,saline, glycerol, sugars and ethanol. Pharmaceutically acceptable saltscan also be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., 18th Edition, Easton, Pa.[1990]).

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. The pharmaceuticalcompositions of the present invention may be manufactured in any mannerknown in the art (e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes).

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding, free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, which is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they may be placedin an appropriate container and labeled for treatment. Foradministration of the activated form of FV or derivativethereof-containing vectors, such labeling would include amount,frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended therapeutic purpose.Determining a therapeutically effective dose is well within thecapability of a skilled medical practitioner using the techniquesprovided in the present invention. Therapeutic doses will depend on,among other factors, the age and general condition of the subject, theseverity of the aberrant blood coagulation phenotype, and the strengthof the control sequences regulating the expression levels of theactivated form of FV or derivative thereof polypeptide. Thus, atherapeutically effective amount in humans will fall in a relativelybroad range that may be determined by a medical practitioner based onthe response of an individual patient to vector-based activated form ofFV or derivative thereof treatment.

D. Administration

The variant activated form of FV polypeptides, alone or in combinationwith other agents may be directly infused into a patient in anappropriate biological carrier as described hereinabove. Expressionvectors of the present invention comprising nucleic acid sequencesencoding the activated form of FV or derivative thereof, or functionalfragments thereof, may be administered to a patient by a variety ofmeans (see below) to achieve and maintain a prophylactically and/ortherapeutically effective level of the activated form of FV orderivative thereof polypeptide. One of skill in the art could readilydetermine specific protocols for using the activated form of FV orderivative thereof encoding expression vectors of the present inventionfor the therapeutic treatment of a particular patient. Protocols for thegeneration of adenoviral vectors and administration to patients havebeen described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699;6,100,242; and International Patent Application Nos. WO 94/17810 and WO94/23744, which are incorporated herein by reference in their entirety.

The activated form of FV or derivative thereof encoding adenoviralvectors of the present invention may be administered to a patient by anymeans known. Direct delivery of the pharmaceutical compositions in vivomay generally be accomplished via injection using a conventionalsyringe, although other delivery methods such as convection-enhanceddelivery are envisioned (See e.g., U.S. Pat. No. 5,720,720). In thisregard, the compositions may be delivered subcutaneously, epidermally,intradermally, intrathecally, intraorbitally, intramucosally,intraperitoneally, intravenously, intraarterially, orally,intrahepatically or intramuscularly. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal applications. A clinician specializing in the treatment ofpatients with blood coagulation disorders may determine the optimalroute for administration of the adenoviral vectors comprising theactivated form of FV or derivative thereof nucleic acid sequences basedon a number of criteria, including, but not limited to: the condition ofthe patient and the purpose of the treatment (e.g., enhanced or reducedblood coagulation).

The present invention also encompasses AAV vectors comprising a nucleicacid sequence encoding the activated form of FV or derivative thereofpolypeptide.

Also provided are lentivirus or pseudo-typed lentivirus vectorscomprising a nucleic acid sequence encoding an activated form of FV orderivative thereof polypeptide

Also encompassed are naked plasmid or expression vectors comprising anucleic acid sequence encoding an activated form of FV or derivativethereof polypeptide.

The Example set forth below is provided to illustrate certainembodiments of the invention. It is not intended to limit the inventionin any way.

Example I

The following materials and methods are provided to facilitate thepractice of the present invention.

Animals

The local Animal Care and Use Committee approved all procedures. Murinemodels for severe FVIII [20] or FIX deficiency [21] were crossed withmice carrying FVL (on C57B16 background) generated by knock-intechnology as previously described [22,23]. The hemophilia B model wasgenerated on a C57B1/6-129 mixed background [21,24] and further crossedinto C57B1/6 for additional five generations. The hemophilia A mice wereon C57B1/6-129 mixed background. Through a series of breedings weobtained hemophilia A or B mice of all three expected FVL genotypes,which allow comparison littermate mice.

Coagulation Assays

Blood samples obtained by tail clipping were collected into 3.8% sodiumcitrate (9 parts of blood: 1 part anticoagulant). Clotting factoractivity was determined by a modified one stage assay incubating 50 μlof human FIX- or FVIII-deficient plasma with 50 μl of automatedactivated partial thromboplastin time (aPTT) reagent (Organon Teknika,Durham, N.C.), and a total of 50 μl of undiluted test sample. Fiftymicroliters of 25 mM CaCl₂ were added, and time to clot formation wasmeasured using Stat4 Coagulation Instrument (Diagnostic Stago,Parsipanny, N.J.). Thrombin-antithrombin (TAT) complexes were measuredby Enzygnost TAT enzyme-linked immunosorbent assay (ELISA) purchasedfrom Dade Behring (Marburg, Germany), as previously described, whichpresent high cross reactivity with murine TAT [25, 26].

Tail Clipping Assay

Mice were anesthetized and the distal portion of the tail (2.5-3 mm ofdiameter) was cut and immersed in 37° C. saline solution. Bleeding timemeasurements exceeding 10 min were stopped by suture of the tail. Theblood loss was determined by measuring the absorbance of hemoglobin(A₅₇₅ nm) in the saline solution in which the tail was placed, asreported [26].

Ferric Chloride (FeCl₃) Carotid Artery Model

The carotid artery of adult mice was exposed, a Doppler flow probe(Model 0.5VB; Transonic Machinery Systems, Ithaca, N.Y.) was placed onthe surface of the exposed artery and a baseline blood flow measurementrecorded. Subsequently, a 2 mm² piece of Whatman #1 paper soaked inferric chloride (15% solution) was applied to the adventitial surface ofthe exposed artery for 2 minutes, after which it was removed, andcarotid artery blood flow recorded. Time to carotid artery occlusion wasdefined as the time after initiation of arterial injury and the onset ofstable occlusion[27].

Real-Time Widefield Intravital Microscope

The cremaster muscle of adult mice was exposed, stretched and pinnedacross the intravital microscopy tray. The rat anti-CD41 (murineplatelet glycoprotein complex IIb/IIIa) Alexa-555 labeled antibody(Molecular Probes, Eugene, Oreg.) was infused at a dose of 10 μg permouse. Immediately after infusion of the antibody, a laser-inducedinjury was performed on the vessel wall of the cremasteric arterioles[28]. The injuries were performed using a pulse-nitrogen dye laserapplied through the micropoint laser system (Phototonic Instruments St.Charles, Ill.). We used an Olympus BX6IWI fixed-stage motorized uprightfluorescence microscope with a long-distance condenser and 40×water-immersion objective. Data analysis was carried out utilizing theSlidebook 4.0 software (Intelligent Imaging Innovations, Denver, Colo.).Fluorescence data were captured digitally up to 10 milliseconds/eventfor 300 frames. The amount of platelet accumulation in the developingthrombi was determined by the sum of all pixel values of theplatelet-specific signal and expressed as relative fluorescence unit(RFU), an arbitrary unit in which the integrated platelet fluorescenceintensity is determined.

Assessing Effects of FV or FVa Proteins in the Hemostasis of HemophiliaMice.

Human FV was isolated from plasma and recombinant FVa was prepared asdescribed before [29]. Purification of both proteins was carried outusing an immunoaffinity column containing anti-human F.V antibody [29].For in vitro activation of FV, 20 nM FV was incubated with murine orhuman thrombin (Haematological Technologies, Inc. Essex-Junction, Vt.)at concentration of 0.25 nM at 37° C. Samples were withdrawn from thereaction mixtures at several time points and the specific cofactoractivity was determined by a PT-based assay using FV-deficient plasma.To determine the cleavage of FV by murine or human thrombin, 300 nM ofsingle chain FV was incubated for several time intervals with thrombin(1 nM). Samples were removed and analyzed by SDS-PAGE. Next, FVa wasinfused via the tail vein into hemophilia B mice and blood samples werecollected by tail clipping prior to protein infusion, and after 15 and120 minutes for determination of FVa levels, aPTTs, and TAT levels.Next, we injected human FVa (30-60 μg/mouse) or FV (60-120 μg/mouse)through the jugular vein of hemophilia A or B mice and monitored clotformation in the intravital microscopy over a period of two hours.

Statistical Analysis

Comparison of data obtained from distinct experimental groups wasanalyzed using JMP version 4.0.2 (SAS Institute Inc. Cary, N.C.).

Results Hemophilia A and B Mice with FVL Present Improved Clotting Times

The determination of clotting activity for hemophilia A mice homozygousor heterozygous for FVL mutation revealed shortening of the aPTT valueswhen compared to hemophilia A mice without FVL (FIG. 1A). Similarimprovement on the APTT values was also determined for hemophilia B micewith FVL (FIG. 1B). We next determined TAT levels, to verify whether theimprovement of the aPTT values was associated with increased thrombingeneration. This immunoassay was developed to detect human TAT but alsopresents high cross-reactivity to murine TAT [25,26]. There was a goodcorrelation between shortening of the aPTT and increased levels of TAT(FIG. 1, panels C and D). However, TAT levels of hemophilia mice withFVL did not reach those of FVL without hemophilia (FIG. 1, panel F).

Blood Loss Following Tail-Clipping is Reduced Among Hemophilia B MiceCarrying FVL.

We next tested whether the mild improvement of the clotting times invitro was associated with in vivo hemostatic performance. Blood loss wasmeasured during a ten minute period after sectioning the distal part ofthe tail. No difference was seen among hemophilia A with or without FVL(FIG. 2A), whereas the blood loss among hemophilia B mice homozygous forFVL was reduced when compared with mice without the mutation (FIG. 2B).

No Sustained Thrombus Formation Following Carotid Artery Injury inHemophilia Mice with FVL.

All normal mice (n=5) or FVL homozygous mice (n=5) tested withouthemophilia presented full vessel occlusion (Table 1), which wascharacterized by the interruption of blood flow within 6 to 8 min postvessel injury. In contrast, no vessel occlusion was detected inhemophilia A mice without FVL (Table 1). In two out of ten hemophilia Amice with FVL, only a transient reduction of the blood flow wasdetected, but not at the levels to suggest full lumen occlusion (Table1). Similar findings were determined among hemophilia B homozygous forFVL, with only one of the eight mice developing a complete and one atransient vessel occlusion.

TABLE 1 Carotid artery occlusion following FeCl₃-induced injury model.N. of N. of mice with occlusion Occlusion Genotype mice TransientComplete time ± SD WT 5 0 5 6.3 ± 2 min FVL (+/+) 5 0 5 6.5 ± 1 min HA 50 0 — HA/FVL (+/−) 5 1 0 — HA/FVL (+/+) 5 1 0 — HB 5 0 0 — HB/FVL (+/−)4 0 0 — HB/FVL (+/+) 8 1 1 6 min WT: wild-type controls; FVL: Factor VLeiden; HA: hemophilia A; HB: hemophilia B.

FVL Restores the Ability to Form Thrombi in Hemophilic Mice at theMicrocirculation Level

We monitored platelet accumulation during real-time imaging of thelaser-induced endothelial damage in the microcirculation. The compositeimage consisted of a brightfield image of the thrombus and fluorescenceimage of platelets (FIG. 3). In normal mice (n=3), we determined thatall injured arteriole sites (n=30) resulted in clot formation. Inaddition, we characterized the thrombus formation of FVL homozygous mice(n=4), and as expected, clots formed in all (n=40) injured sites (Table2).

TABLE 2 Summary of thrombus formation following laser-inducedendothelial damage. Genotype N. of mice N. of sites injured N. of clots(%) WT 3 30 30 (100) FVL 4 40 40 (100) Hemophilia A FVL (+/+) 5 45 45(100) FVL (+/−) 6 43 43 (100) FVL (−/−) 8 39 0* F.VIII infusion 3 8  8(100) Hemophilia B FVL (+/+) 4 40 40 (100) FVL (+/−) 3 18 18 (100) FVL(−/−) 4 16 0* F.IX infusion 3 21 21 (100) Infusion of FVa Hemophilia A 28  8 (100) Hemophilia B 2 13 13 (100) Infusion of FV Hemophilia A 2 100* Hemophilia B 2 11 0  WT: wild-type controls; FVL: Factor V Leiden;(+) Denotes presence of FVL mutation. For infusion experiments, purifiedF.IX (Monomine, Aventis Behring, Kankakee, IL) and recombinant F.VIII(Kogenate FS, Bayer, West Haven, CT) were used. *Fischer's exact testwas used for statistical analysis comparison between hemophilic micewith and without FVL (P < 0.001) or mice infused with FV and FVa (P <0.001).

In contrast, no thrombus formation was detected in mice with hemophiliaA (n=8) or B (n=4) following successive vascular injuries to a total of68 injury sites, averaging 3-10 sites per mouse (FIG. 3, panel A; Table2). However, when these animals received intravenous injection of humanpurified FVIII or FIX concentrates at doses to achieve ˜100% of normallevels, clots formed in all injury sites (FIG. 3, panel B).

We next tested hemophilia A (n=5) or B mice (n=4) homozygous for FVL.Clot formation was observed in all 85 injured sites (ranging from 5-10sites/mouse) and remained stable for the duration of the experiment Wig.3 panels E and F). Interestingly, similar results were obtained inhemophilia A (n=6) or hemophilia B (n=3) mice heterozygous for FVL. Inthis group, a total of 61 injury sites were analyzed and thrombusformation was consistently detected (FIG. 3, panel D). Time course ofplatelet deposition is shown in FIG. 4 for groups of hemophilic mice.The data is represented by the median values of RFU derived from theplatelet deposition, which represent a relative comparison amongdifferent groups tested. There was a increase in platelet depositionover time for both hemophilia models with FVL as compared to hemophiliamice without the mutation (FIG. 4, panel B).

Infusion of Purified FVa Induces Thrombus Formation in Murine Models ofHemophilia A or B

Initially we have determined that murine thrombin activates human FV ina similar fashion to human thrombin. To test if transient increment inthe FV or FVa levels could mimic the in vivo effect of FVL, we injectedmice with purified human FV or FVa. Because FVa is secreted in theactive form, there is no requirement for thrombin activation of thepurified protein [29]. Therefore, direct interpretation of these data isnot confounded by traces of thrombin in the protein solution infused.Infusion of 30 μg of FVa/mouse into hemophilia B mice resulted indiscrete shortening of the aPTT from 77±3 seconds at baseline to 72±4seconds at both time points (15 or 120 minutes) post infusion. Thesedata were in good agreement with increased levels of TAT from 15±5 ng/ml(at baseline) to 69±10 and 67±22 ng/ml at 15 and 120 minutes,respectively. We next monitored clot formation in the intravitalmicroscopy. Prior to the protein infusion, no clot formation wasobserved in both hemophilia A and B mice, as expected. Followinginfusion of 30 μg or 100 μg of FVa, platelet accumulation was readilyobserved at all injury sites (FIG. 4, panel C and D). In contrast, whenmice were injected with the procofactor FV at comparable doses toachieve similar plasma concentration (0.180 or 0.360 pmoles), no clotformation was detected. Collectively these results were similar for bothhemophilia models (Table 2 and FIG. 4, panels C-D).

Discussion

The assessment of the clinical impact of FVL on the hemophilia phenotypehas been controversial and hampered by the complex interaction of otheracquired and genetic modifying factors. Therefore, the use of murinemodels minimizes the influence of several acquired factors. Theoccurrence of spontaneous bleeding episodes in murine models for severehemophilia A or B is rare. Thus, to properly address the effect of FVLon the severe hemophilia phenotype, a series of in vivo hemostatic testswere imposed upon these animal models.

Hemophilia mice with FVL mutation presented improvement in hemostasis,as determined by shortening of the aPTT-based assays and increased TATlevels, which reflects enhanced thrombin generation. To address whetherimprovement on in vitro parameters of hemostasis had any relevant impacton the hemophilia phenotype in vivo, mice underwent a series ofhemostatic challenges.

In one model a mechanical injury was induced by transfection of the tailvessels; in a second, the injury was induced by a ferric chloridechemical to the carotid artery. The latter model is characterized byoxidative injury that disrupts the endothelium and exposes thesubendothelium [30]. Hemophilia B mice homozygous for FVL presentedmodest improvement of hemostasis in both methods when compared withlittermates without FVL whereas among hemophilia A models no improvementwas found. It is possible that differences in mouse strains may affectsome of the hemostatic parameters, as already shown for FVL [22]. Thesedata suggest that, upon injury of large vessels, FVL does not provide amajor beneficial hemostatic effect.

The real-time imaging of thrombus formation developed by Furie andcolleagues [28], in conjunction with the laser-induced endothelialinjury model [31], provides a sensitive method for evaluation ofhemostasis at the microcirculation of a living animal. It has beenobserved that the outcome of this method is not full occlusion of thevessel lumen; rather, endothelial cells appear to be activated insteadof disrupted with less exposure of the subendothelium [28,31]. Inhemophilia mice with no FVL mutation, no clot was observed even uponsuccessive laser expositions. However, following replacement of themissing clotting factor by intravenous infusion of human FVIII or FIXconcentrates, clot formation was observed in all arteriole sitesinjured. Hemophilia A or B mice homozygous for the FVL mutation haverestored ability to form a thrombus. Interestingly, among hemophiliamice heterozygous for FVL, thrombus formation is comparable tohomozygous FVL. This is particularly interesting since most humansubjects with hemophilia and FVL carried only one FVL allele.

When extrapolating data from murine models to humans it is important toconsider the differences between human and murine FV such the origin ofFV synthesis [32,33] and differences in the APC pathway such as theability of human aPC to properly inactivate murine FVa [34], and theabsence of the plasma protein C inhibitor [35]. To better assess therole of the APC pathway as a modifier of the hemophilia phenotype, othercomponents such as thrombomodulin or endothelial protein C receptor(EPCR) functions [36] need to be investigated. The vascular distributionof thrombomodulin and EPCR differs as a function of vessel size. Theresults of murine models are informative because improvements inhemostasis by FVL seem to be vessel-dependent (i.e. micro vs. macrocirculation). Thrombomodulin concentration in the microcirculationis >1000-fold higher than in vessels of −0.3 cm diameter, whichfacilitates binding to thrombin and consequently clotting activity isdepressed. We speculate that in the presence of FVL, the continuousthrombin generation at the microcirculation may lead to high freethrombin; therefore, the coagulation is locally enhanced. In largevessels, such as the murine carotid artery (diameter of ˜0.55 mm),thrombin is likely free since thrombomodulin levels are relatively low[37,38]. Therefore, the impact of the FVL is not sufficient tosignificantly alter hemostasis at the macrocirculation level.

Using human hemophilia A plasma, Mann and colleagues demonstrated thatslow formation of FVa is an additional factor that impacts the impairedthrombin generation. Because free FXa is rapidly inactivated, thepresence of fully active FVa complexed with FXa in the prothrombinasecomplex is critical to prevent FXa inactivation by antithrombin andtissue factor pathway inhibitors [39]. Further experiments showed thatincreasing FV levels up to 150% in hemophilia A plasma did not result insignificant enhancement of thrombin, whereas adding FVL to levels of100% or 150% enhanced thrombin generation by 3 and 5-fold, respectively[1,5]. Recently, a similar effect was determined in hemophilia B plasma[1,6]. Therefore, we hypothesized that an increase in FVa levels couldmimic the effect of FVL in hemophilia mice. The results demonstratedthat FVa, but not FV, has the ability of restoring the hemophiliaphenotype at the microcirculation level, in a similar manner as findingsindicate that FVL does.

There is evidence that in humans FV, but not FVL, presents cofactoractivity in the APC-mediated inactivation of FVIIIa. Recently, Simioniet al demonstrated increased thrombin generation and risk for venousthrombosis among homozygous FVL and heterozygous for FVL with partial FVdeficiency (pseudo-homozygous APC resistance) when compared to subjectsgenotyped as heterozygous for the FVL mutation (wild-type FV activitypreserved) [40]. Here we found that littermate hemophilic miceheterozygous and homozygous for FVL presented improvement of both invitro and in vivo hemostatic parameters at similar fashion. These datasuggest that it is slow inactivation of FVa with consequentlyprocoagulant activity that underlying the potential benefits of FVL inhemophilia. Therefore, it is possible that murine FV does not presentcofactor activity on FVIIIa-inactivation by APC. Although theseexperimental conditions may present distinct underlying mechanisms, dataobtained in both genetic models and protein injection demonstrate thebeneficial effects of the enhancement in thrombin levels, as the finaloutcome, and suggest that other alternative therapeutic approaches forhemophilia could be investigated.

In summary, this work demonstrated that the FVL mutation enhanceshemostasis in hemophilia A or B mice as judged by the improvement of theclotting times and by the in vivo ability to form clots. Because FVL hasmarkedly beneficial effects at the microcirculation level in an injurythat predominantly does not expose the subendothelium, the protectionagainst hemorrhagic challenges are likely to impact minor bleedings, butnot those trauma-induced hemorrhages. These data explain in part theheterogeneity of the clinical diversity observed for the severehemophilia phenotype with FVL especially among adults.

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method for the treatment of hemophilia in a patient in needthereof, comprising administering an effective amount of an activatedform of FV variant or a derivative thereof, thereby enhancing clotformation in said patient and ameliorating the symptoms of acquired andinherited bleeding disorder.
 2. The method of claim 1, wherein saidderivative is selected from the group consisting of FV-810; factor Vlacking amino acids 811-1491, FV-859; factor V lacking amino acids860-1491; FV-866; factor V lacking amino acids 867-1491; FV-902; factorV lacking amino acids 903-1491; FV-924; factor V lacking amino acids923-1491; FV-937; factor V lacking amino acids 938-1491; FV-956; factorV lacking amino acids 957-1491; FV-1033-B58-s131; factor V lacking aminoacids 1034-1491 with amino acids 900-1030 exchanged with amino acids907-1037 of factor VIII; FV-1033-B58-s104; factor V lacking amino acids1034-1491 with amino acids 904-1007 exchanged with amino acids 972-1075of factor VIII; and FV-1033-B58-s46; factor V lacking amino acids1034-1491 with amino acids 963-1008 exchanged with amino acids 1032-1077of factor VIII.
 3. The method of claim 1, for the treatment ofhemophilia A.
 4. The method of claim 1, for the treatment of hemophiliaB.
 5. The method of claim 1, wherein said activated form of FV orderivative thereof is delivered intravenously.
 6. The method of claim 1,wherein a vector encoding the activated form of FV or derivative thereofis administered to said patient.
 7. The method of claim 1, wherein saidvariant is a pro-coagulant and said disorder is selected from the groupconsisting of hemophilia A and B, hemophilia A and B associated withinhibitory antibodies, coagulation factor deficiency, vitamin K epoxidereductase C1 deficiency, gamma-carboxylase deficiency, bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation treatment disorders, Bernard Soulier syndrome,Glanzman thromblastemia, and storage pool deficiency.
 8. The method ofclaim 7, wherein said over-anticoagulation treatment disorder resultsfrom administration of heparin, low molecular weight heparin,pentasaccharide, warfarin, small molecule antithrombotics and FXainhibitors.
 9. The method of claim 1, wherein said variant isadministered intravenously at least once a day at a dosage between about10 and 500 μg/kg.
 10. An isolated nucleic acid molecule encoding anactivated form of FV, wherein said activated form of FV is selected fromthe group consisting of FV-859, FV-866, FV-924, and FV-937.
 11. Anisolated activated form of FV, wherein said activated form of FV isencoded by the nucleic acid molecule of claim
 10. 12. An isolatednucleic acid molecule encoding an activated form of FV, wherein at leastone of the arginine residues at positions 506, 306, and 679 of saidactivated form of FV is replaced with an amino acid other than arginine.13. The nucleic acid molecule of claim 12, wherein the arginine residuesare replaced with a glutamine.
 14. The nucleic acid molecule of claim12, wherein said activated form of FV is selected from the groupconsisting of FV-810; FV-859; FV-866; FV-902; FV-924; FV-937; FV-956;FV-1033-B58-s131; FV-1033-B58-s104; and FV-1033-B58-s46.
 15. An isolatedactivated form of FV, wherein said activated form of PV is encoded bythe nucleic acid molecule of claim 12.